The solid-phase peptide synthesis (SPPS) of biologically active peptides, glycopeptides and glycoconjugates is a field of great interest. While SPPS is generally a mature technology, there is still an urgent need for improved methods to synthesize “difficult” peptides and peptide conjugates. Various peptides and/or conjugates are considered “difficult” when the synthesis of the peptide sequences results in incomplete peptide bond formation and/or when deprotection reactions occur at various stages in the SPPS.
These synthesizing problems can result from steric effects when one or both amino acid units at the newly formed amide bond are bulky or possess β-branched side chains such as valine, isoleucine, threonine. Similarly, the problems can occur at glycosyl amino acids which are found in glycopeptides.
These problems occur with most of the common peptide coupling reagents and are more pronounced in solid-phase peptide synthesis due to the steric requirements of the resins used in the synthetic process. Attempts to overcome these problems include the use of larger molar equivalents of peptide coupling reagents and amino acids, or the use of repeated cycles of coupling, washing and recoupling. However, the use of additional reagents and time increases the cost of materials and the time required to complete the synthesis.
Other attempts to synthesize difficult or long peptides include the use of a mixed-phase synthesis process. A mixed phase synthesis includes the production of peptide fragments through solid-phase peptide synthesis, followed by an assembly of these fragments in solution. However, if the peptide coupling is not complete, either on the solid phase or in solution, then closely related peptide-based impurities will be introduced into the peptide synthesis process. This is of special concern since peptides of very high purity (>95 area-% by HPLC) are often required for use in medical applications. In addition, removing these closely related impurities can be difficult or impossible. Also, the additional purification effort adds to the cost of the overall peptide synthesis.
As one example of a difficult synthetic process, as described by Krüger, et al. (Eur. J. Org. Chem. 2008, 35, 5936-5945, in the linear solid-phase synthesis of a 31-mer peptide being investigated for the treatment of diabetes, fifteen (15) coupling steps were needed due to incomplete coupling. Further, the unreacted terminal amino groups that were formed had to be capped by acetylation.
As a consequence, others have used a mixed-phase synthesis as a strategy to deal with the difficult sequences in SPPS. In fact, currently, most large scale peptide syntheses of peptides longer than five amino acids are generally manufactured by a mixed or a convergent strategy (i.e., synthesis of small segments or fragments that are subsequently joined to give the final sequence). The mixed strategy, being convergent in nature, consumes less raw materials and reagents. Still, the overall yield of a synthesis becomes critical when considering that many pharmaceutically related peptides must be prepared on a multi-ton scale. Thus, there is an ongoing search for and selection of new starting reagents that enable the production of peptides faster, in a safer way, and at lower costs (Riniker, et al. Tetrahedron 1993, 49, 9307-9320; Barlos, et al. Liebigs Ann. Chem. Ann. Chem. 1993, 215-220; Bray Nat. Rev. Drug Discovery 2003, 2, 587-593; Andersson, et al. Biopolymers 2000, 55, 227-250; Bruckdorfer, et al. Curr. Pharm. Biotechnol. 2004, 5, 29-43).
Another technology to deal with difficult peptide synthesis is microwave irradiation during peptide synthesis (Coin, et al. Nature Protocols, 2007, 2, 3247-3256).
Still another technology includes the use of fluorous tag reagents which allow for solution phase synthesis and solid phase extraction of fluorous tag (Chen and Zhang, Organic Letters 2003, 5, 1015; Marshall and Liener, J. Org. Chem. 1970, 35, 867; Curran, D. P. Synlett 2001, 1488). The latter offers advantages common to solution phase chemistry and maintains the rapid nature of solid phase supports (Zhang, ACS Symposium Series 949, 2007, 207-220).
In addition to synthesizing peptides, there is a need to synthesize a variety of peptide conjugates. The purpose of creating a peptide conjugate can be manifold. One of the most well recognized and medicinally important types of peptide conjugate include PEGylated peptides and proteins. PEGylation is the practice of covalently coupling of poly(ethylene glycol) by the use of a PEGylation reagent to pharmaceutical proteins and peptides to improve their pharmacological properties. The original method was described by Davis and Abuchowski in the 1970s (Abuchowski et al. J. Biol. Chem. 1977, 252, 3582-3586). PEGylation has become the dominant protein/peptide-based drug delivery system for the biotech industry, with sales of PEGylated peptide and protein-based drugs reaching over $4 billion (Maggon, in Handbook of Pharmaceutical and Biotechnology, “R&D paradigm shift and billion-dollar biologics”, John Wiley, 2007, pp. 161-198). Brocchini et al. (Adv. Drug Delivery Rev. 2008, 60, 4-13) noted that one of the key issues facing the field of PEGylation is the need to obtain site-specific peptide or protein PEGylation to avoid the loss of biological activity. It has also been noted that the desirable properties of PEGylation are not exclusive for poly(ethylene glycol) (Veronese Adv. Drug Delivery Rev. 2008, 60, 1-2).
For example, polymers of origin such as polysaccharides (Gregoriadis, et al. Int. J. Pharm. 2005, 300, 125-130; Fernandes, et al. Int. J. Pharm. 2001, 217, 215-224) and synthetic polymers (Veronese, et al., in: M. J. Harris, S. Zalipski (Eds.), Poly(ethylene glycol) Chemistry and Biological Applications, ACS Symposium Series, vol. 680, 1977, pp. 182-192; Miyamoto, et al., Macromolecules 1990, 23, 3201-3205; Gaertner, et al. J. Control. Release 2007, 119, 291-300) have demonstrated that molecules other than poly(ethylene glycol) can favorably modify protein and peptide properties.
To address both the need for new methods for difficult peptide synthesis and the need for site specific peptide and protein conjugation methods, new amide bond forming reactions are being researched. One proposed method included the reaction of thioacids with 2,4-dinitrobenzenesulfonamides (dNBSs). Originally, the dNBS group was used as a protecting group for the alkylation of primary amines (Fukuyama, et al. Tetrahedron Lett. 1997, 38, 5831-5834).
The dNBS group as a N-sulfonamide derivative 1 (FIG. 26—PRIOR ART Scheme 1) was later found to facilitate the formation of amides 3 in the presence of thioacids (Messeri, et al. Tetrahedron Lett. 1998, 39, 1669-1672; Messeri, et al. Tetrahedron Lett. 1998, 39, 1673-1676). The mechanism involves ipso attack of the thioacid on the sulfonamide to form a Meisenheimer complex 2. The nitrogen of the sulfonamide ultimately attacks the adjacent thioester with extrusion of SO2. The chemistry has been shown to be amenable to the coupling of hindered amino acid derivatives to form native peptide fragments and peptide conjugates (Crich, et al. Org. Lett. 2007, 9, 4423-4426, Crich, et al. Org. Lett. 2007, 9, 5323-5325).
In addition the β-N-dNBS-glycosylsulfonamides have been shown to provide β-configured glycosyl amino acids common to most N-linked glycopeptides/proteins when reacted with amino thioacids (Talan et al. Carbohydr. Res. 2009, 344: 2048-2050). The reaction between dNBS-modified amines and thioacids can be performed at room temperature and is complete in thirty minutes. The observed yields of this ligation are comparable with other methods such as the Staudinger ligation reaction (Doores et al. Chem. Comm. 2006, 1401-1403; He et al. Org. Lett. 2004, 6, 4479-4482; Györgydeák, et al. Bioorg. Med. Chem. 2004, 12, 4861-4870).
Aside from the efficient chemistry underlying a given amide bond forming reaction, its adoption depends on the straightforward synthesis of the requisite fragments to be coupled. Some, N-peptidyl arylsulfonamides have been reported to be amenable to SPPS synthesis. For example, ortho-nitrobenzenesulfonamides (o-NBS) and paranitrobenzenesulfonamides (p-NBS) have been coupled to the N-terminus of a peptide bound to Rink amide 4-methylbenzhydrylamine (MBHA) resin (Miller, et al. J. Am. Chem. Soc. 1997, 119, 2301-2302) while o-NBS-amino acids have been synthesized off-resin and used in SPPS (Miller, et al. J. Am. Chem. Soc. 1998, 120, 2690-2691). In both instances, the N-arylsufonyl group was used as protecting group to aid in the controlled alkylation of the N-terminus, and was removed afterwards.
Consequently, there is a need in the field of peptide synthesis and peptide conjugate synthesis for chemistry capable linking sterically demanding amino acids to form difficult peptides.
Furthermore, there is a need for linking chemistry that can facilitate the sitespecific conjugation of an untold number of compounds to peptides and proteins, in particular PEGylating reagents, polymers, and saccharides.
Despite the diverse methods describe above, there is still a need in the field of peptide synthesis and peptide conjugate synthesis for a synthetic method that is capable of linking sterically demanding amino acids to form difficult peptides.
Furthermore, there is a need for a synthetic method that can facilitate the site-specific conjugation of one or more types of desired compounds to peptides and proteins, such as, in particular PEGylating reagents, polymers, and saccharides.
The inventors herein have now greatly improved upon previous methods for peptide synthesis.